We have observed the induction by mutagen treatment of colonies of Chinese hamster ovary (CHO) cells which are sectored (mosaic) in appearance when stained for glucose-6-phosphate dehydrogenase (G6PD) activity. This phenomenon occurred when populations of single CHO cells were exposed to the mutagen ethyl methane sulfonate, (EMS), allowed to grow into colonies and stained histochemically for G6PD activity. Colonies composed of unstained and stained cells in various sectored patterns and uniformly unstained colonies (pure mutant) were observed at a frequency of about 1 per 1000 colonies. The influence of the replication state of the G6PD gene on the sectoring process was examined using synchronized cell populations which were exposed to the mutagen in various parts of the cell cycle. Cells exposed in G1 to UV light and EMS gave rise to colonies mosaic for G6PD activity at a frequency comparable to an asynchronous culture. Cell aggregation is not a significant cause of the mosaic phenomenon. G6PD-cells isolated from mosaic colonies yield stable populations of G6PD- colonies.